As a nucleoside synthesis-related enzyme, nucleoside hydrolase II is known to be involved in the degradation of nucleoside and 5′-nucleotidase is known to be involved in the nucleoside synthesis.
A gene encoding Nucleoside hydrolase II is the gene encoding the enzyme catalyzing ribose and corresponding bases, which are purine and pyrimidine, irreversibly, in salvage pathway of nucleoside. This gene contains a 1326 bp nucleoside sequence and encodes a protein of 308 amino acids and has substrate-specificity to both of purine and pyrimidine nucleosides (Microbiology, 2006, vol. 152, p 1169-1177).
A gene encoding 5′-nucleotidase is the gene encoding the enzyme catalyzing dephosphorylation of nucloeside such as AMP, GMP, IMP and XMP in nucleoside biosynthesis pathway (de novo pathway). This gene encodes a protein of 705 amino acids.
Inosine is an important substrate for the chemical synthesis and synthesis by enzyme transfer of 5′-inosinic acid which is in the lime light as a flavor-enhancing seasoning. Also, inosine is an intermediate of nucleic acid biosynthesis, which is not only an important physiological factor in animals and plants but also applied in various fields including food and medicinal industries. Nucleoside and its derivatives have been reported to have many usages as antibiotic, anti-cancer and anti-viral substances (J. Virol, vol; 72, pp 4858-4865).
The conventional method to produce inosine is direct fermentation using such microorganism as Bacillus (Agric. Biol. Chem., 46, 2347 (1982); Korean Patent No. 27280) or Corynebacterium ammoniagenes (Agric. Biol. Chem., 42, 399 (1978)), or thermal decomposition of 5′-inosinic acid (Japanese Laid-off Patent Publication No. S43-3320). However, the thermal decomposition requires massive heat, which makes it not practical. According to the direct fermentation, activity of the strain producing inosine is very low, so that production cost increases and fermentation takes longer, resulting in the low productivity.
To produce inosine by direct fermentation using a microorganism at high concentration with high yield, it is very important to develop a strain that is appropriate for the accumulation of inosine at high concentration in its culture broth.
The present inventors studied persistently to produce inosine with high productivity/high yield by direct fermentation using a microorganism. And at last, the present inventors completed this invention by confirming that the microorganism producing 5-inosonic acid could produce inosine with high productivity/high yield when rih II gene encoding nucleoside hydrolase II is inactivated and ushA gene encoding 5′-nucleotidase is enhanced.